Algal Research 11 (2015) 284293

Contents lists available at ScienceDirect

Algal Research
journal homepage: www.elsevier.com/locate/algal

Identication and mechanism analysis of chemical modulators

enhancing astaxanthin accumulation in Haematococcus pluvialis
Xinheng Yu, Xiangfeng Niu, Xiaoqing Zhang, Guangsheng Pei, Jing Liu, Lei Chen , Weiwen Zhang
Laboratory of Synthetic Microbiology, School of Chemical Engineering & Technology, Tianjin University, Tianjin 300072, PR China
Key Laboratory of Systems Bioengineering, Ministry of Education of China, Tianjin 300072, PR China
Synbio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin, PR China

a r t i c l e

i n f o

Article history:
Received 31 March 2015
Received in revised form 12 June 2015
Accepted 11 July 2015
Available online xxxx
Keywords:
Chemical modulators
Astaxanthin
H. pluvialis
Metabolomics

a b s t r a c t
Haematococcus pluvialis is one natural source for high-value anti-oxidant astaxanthin. In this study, we evaluated
23 chemicals of 8 groups (antioxidant, oxidant, signal transducer, metal ion, auxin, gibberellin, cytokinin and
amine) for their effects on astaxanthin accumulation in H. pluvialis. The results showed that oxidant and signal
transduction chemicals increased astaxanthin accumulation in H. pluvialis signicantly by more than 20%. To reveal possible regulatory mechanism, a gas chromatographymass spectrometry (GCMS) and a liquid chromatographymass spectrometry (LCMS) based metabolomic analysis were applied to determine metabolic
proles of H. pluvialis treated with chemical modulators, and then the metabolomic data was analyzed by a partial least squares-discriminate analysis (PLS-DA) and a weighted correlation network analysis (WGCNA) to identify possible metabolic modules and metabolites relevant to the increased astaxanthin accumulation. The
analyses showed six metabolic modules and seven hub metabolites were possibly related to the stimulatory
roles of oxidant or signal transduction in H. pluvialis. The study provided new insights to the regulatory mechanism of oxidant and signal transduction chemicals on astaxanthin accumulation in H. pluvialis.
2015 Elsevier B.V. All rights reserved.

1. Introduction
The carotenoid astaxanthin (3,3-dihydroxy-,-carotene-4,4dione) has been found in microalgae, aquatic animals, birds and yeast
and is a high-value pigment widely applied in an abroad range of
areas, such as aquaculture and the nutraceutical, pharmaceutical, and
cosmetic industries due to its high anti-oxidative activity [1,2].
Haematococcus pluvialis can accumulate up to 56% (w/w) of
astaxanthin on dry weight basis as a self-defense mechanism under various stresses, thus has become an important natural resource of
astaxanthin [3,4]. Signicant researches have been conducted in
H. pluvialis to enhance microalgal biomass production [5,6], and to understand the regulation of carotenogenesis related to astaxanthin biosynthesis [79], especially astaxanthin accumulation in response to
various stress conditions, such as nutrient stress, excess sodium acetate
along with iron, salinity, light intensity and elevated temperature [8,10,
11]. Results showed that accumulation of astaxanthin in H. pluvialis is
triggered when the cells are exposed to various stresses [12].
In addition to various efforts to improve growth and astaxanthin accumulation [5,6,13], as an alternative approach, chemicals, which

Corresponding authors.
E-mail addresses: lchen@tju.edu.cn (L. Chen), wwzhang8@tju.edu.cn (W. Zhang).

http://dx.doi.org/10.1016/j.algal.2015.07.006
2211-9264/ 2015 Elsevier B.V. All rights reserved.

served as metabolic triggers or enhancers that are able to directly adjust

cellular metabolism, could be applied in H. pluvialis to improve production or accumulation of astaxanthin [14,15]. For example, reactive oxygen species (ROS), such as H2O2, 2,2-azo-bis(2-amidinopropane)dihydrochloride (AAPH) for peroxy radical (AO2), methylene blue
(MB) for singlet oxygen (1O2), and methyl viologen (MV) for superoxide
anion radical (O
2 ), were demonstrated to induce astaxanthin accumulation to about 50 pg/cell in H. pluvialis[16]; more recent studies showed
that astaxanthin accumulation could be enhanced by jasmonic acid
(JA), salicylic acid (SA), gibberellic acid (GA3), abscisic acid (ABA) and
2,4-epibrassinolide (EBR); and the enhancing effects of the chemicals
were concentration-dependent [1720].
To determine molecular mechanisms related to astaxanthin biosynthesis and regulation in H. pluvialis, global analytical approaches, such as
transcriptomics [21,22] and proteomics [23,24], have been employed.
Metabolomics, which shows the concentration level of metabolites,
has been recently applied as a powerful tool to describe the comprehensive metabolic status in biological samples and reveal the dynamic metabolic response to the exogenous or endogenous disturbance [25,26]. In
a previous study, we had applied metabolomics to study astaxanthin
accumulation under various stress conditions in H. pluvialis, and the results showed that L-glutamic acid (GLU), -ketoglutaric acid (AKG) and
D-ribose

5-phosphate (R5P) are possible metabolites associated with

astaxanthin biosynthesis, demonstrating that metabolomic analysis is

X. Yu et al. / Algal Research 11 (2015) 284293

a powerful tool to nd possible metabolic precursors for bioproducts

[11].
In this study, to identify new chemical modulators capable of enhancing astaxanthin accumulation in H. pluvialis, we selected 23 chemical modulators, including antioxidant, oxidant, signal transducer, metal
ion, auxin, gibberellin, cytokinin and amine, according to their stimulatory roles on other bioproducts in various microalgae [14,27] and then
conducted a phenotypic screening for their roles on astaxanthin accumulation. Our results indicated that oxidant and signal transducer induced a signicant increase in astaxanthin accumulation in H. pluvialis.
In addition, a combination of chromatographymass spectrometry
(GCMS) and liquid chromatographymass spectrometry (LCMS)
based metabolomic analysis was applied to obtain the metabolic proles of H. pluvialis treated with various chemical modulators and utilized and metabolomic datasets to construct a metabolic network
using a weighted correlation network analysis (WGCNA) to illustrate
metabolic modules and hub metabolites signicantly associated with
each of the chemical modulators [28,29]. The analysis reveals possible
metabolic biomarkers and regulatory modules related to effects of
chemical modulators on astaxanthin biosynthesis, which will be valuable in deciphering regulatory mechanism of astaxanthin biosynthesis
in H. pluvialis and discovering or even designing new chemical modulators to further enhance astaxanthin accumulation.

285

2.3. Astaxanthin extraction and determination

Astaxanthin extraction from H. pluvialis was conducted according to
previous publications [31,32]. At the early stage in 24 well plates,
astaxanthin was analyzed using a ow cytometer (FACSCalibur,
BectonDickinson, San Jose, CA, USA) equipped with an ion argon
laser (excitation 488 nm, 30 mW power). FL2, emitted at 582
21 nm, was used to detect astaxanthin uorescence. For the ow cytometric measurements, 20,000 cells were acquired. At the later stage in
a shake ask, the astaxanthin content was analyzed using a high performance liquid chromatography (HPLC) equipped with a Symmetry C18
column (5 m 4.6/250 mm) (Waters, Milford, MA, USA) and with an
isocratic solvent system consisting of dichloromethane:acetonitrile:
methanol (20:70:10, v/v/v) at a ow rate of 0.7 mL/min [33]. The
HPLC consisted of a 1260 series device (Agilent Technologies,
Waldbronn, Germany) with a diode array detector (476 nm). The
injection volume was 10 L. We determined the astaxanthin and biomass in different samples, calculated the volumetric productivities of
astaxanthin (mg astaxanthin/L liquid), and then normalized the data
by setting control as 100%. To validate differences of astaxanthin contents between the treated and the control samples, a statistical t-test
model was applied for the comparative analysis, and p-values of less
than 0.05 were considered statistically signicant [27,34].

2. Materials and methods

2.4. Metabolomic analysis
2.1. Chemicals
H2O2, MgCl2, CuSO4 and ZnSO4 were purchased from Kewei Inc. (Tianjin,
China), methylene blue (MB) and 2,2-azo-bis(2-amidinopropane)dihydrochloride (AAPH) were purchased from Heowns Inc. (Tianjin,
China), 3-iodoacetate acid (IAA), 3-indolebutyric acid (IBA), abscisic
acid (ABA), zeatin (ZA) and kinetin (KT) were from Solarbio Inc.
(Beijing, China), butylated hydroxyanisole (BHA) and propyl gallate
(PG), 1-naphthalene acetic acid (NAA), 2,4-dichlorophenoxy acetic
acid (2,4-D) and gibberellic acid (GA3), methyl viologen (MV),
naphthoxyacetic acid (BNOA), 2-chlorodracylic acid and ethanolamine
(ETA) were purchased from Sigma-Aldrich (St. Louis, USA), salicylic
acid (SA) was purchased from Sangon Inc. (Shanghai, China), jasmonic
acid (JA) was purchased from Tokyo Kasei Inc. (Tokyo, Japan), and 2,4epibrassinolide (EBR) was purchased from Medchem Express Inc.
(Princeton, USA). All chemicals used are analytically pure. JA, ABA,
EBR, IAA, IBA, NAA, 2,4-D, BNOA, 2-chlorodracylic acid, ZA and KT
were dissolved in dimethyl sulfoxide (DMSO), GA3 was dissolved in
ethanol, and others were dissolved in double-distilled water. BHA, PG,
ABA, ZA, KT and EBR were made in amber glass vials and stored in the
dark at 20 C. Others were made in amber glass vials and stored in
the dark at 4 C.

All chemicals used for metabolome isolation, GCMS and LCMS

analyses were obtained from Sigma-Aldrich (Taufkirchen, Germany).
For metabolomic analysis, cells were collected by centrifugation at
8000 g for 10 min at 4 C (Eppendorf 5430R, Hamburg, Germany) at
two time points (i.e., 6 d and 9 dafter inoculation). The cell pellets
were immediately frozen in liquid nitrogen and then stored at
80 C. LCMS, GCMS and WGCNA metabolomic analyses were
preformed according to our previous publications [35,36]. LCMS
metabolomic prole data was rst normalized by the internal control
and the cell numbers of the samples, and then subjected to partial
least squares-discriminate analysis (PLS-DA) using software SIMCA-P
11.59 and heatmap analysis using software MEV 4.9.0. PLS-DA and
WGCNA were applied to analyze the GCMS metabolomic prole data
normalized by the internal control and the cell numbers of the samples.
For WGCNA, the modules with correlation r N 0.5 or r b 0.5 and statistical signicance p-value of less than 0.05 were extracted for further
investigation. Hub metabolites were screened by high connectivity
with other metabolites (5) in the modules strongly associated with
phenotype.

2.5. Pathway enrichment analysis

2.2. Microalgal strain and cultivation
H. pluvialis NIES 144 was obtained from the National Institute for Environmental Studies in Tsukuba, Japan. It was grown on the medium as
described previously [11]. At the early stage, the cells were cultivated in
24 well plates each containing 2 mL medium. At the later stage, the cells
were grown in 250 mL Erlenmeyer asks each containing 100 mL of
medium. Cultures were incubated in a growth chamber at 22 C under
continuous light of 20 mol photons m 2 s 1. The ask was shaken
manually once a day [30]. The seed cultures were cultivated for 3 d
after reaching exponential growth phase, and then were used to inoculate the fermentation asks at an inoculum size of 10% (v/v). All chemical modulators involved were added at 3 d after inoculation. We
established three control experiments in parallel with the chemicalspiked cultures, the rst with 0.1% DMSO (v/v), the second with 0.1%
ethanol (v/v) and the third with sterilized double-distilled water.
Three biological replicates were established for each concentration.

Pathway enrichment analysis was preformed according to our previous publications [11]. Briey, the protocol was conducted according to
KEGG (Kyoto Encyclopedia of Genes and Genomes) Database using
the following formula:
 NM 
 ni
 :

m1
X M
i

P 1

i0

N
n

N is the number of all metabolites with all KEGG pathways, M is the

number of metabolites with a given KEGG pathway, n is the number of
the associated metabolites with all KEGG pathways, and m is the number of the associated metabolites with a given KEGG pathway. The
KEGG pathways with a p-value of less than 0.05 were considered as
enriched KEGG pathways.

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X. Yu et al. / Algal Research 11 (2015) 284293

3. Results and discussion

3.1. Screening of chemical enhancers for astaxanthin accumulation
Based on previous studies of chemicals on bioproduct accumulation
in a diverse of microalgae [14,27], 23 chemicals from 8 chemical groups,
including antioxidant, oxidant, signal transducer, metal ion, auxin, cytokinin, gibberellin and amine were selected for evaluation of their effects
on astaxanthin accumulation in H. pluvialis (Table 1). Since GA3 was dissolved in ethanol, while BHA, PG, JA, ABA, EBR, IAA, IBA, NAA, 2,4-D,
BNOA, 2-chlorodracylicacid, and KT were dissolved in DMSO, three
controls were established: one with 0.1% DMSO (v/v), another with
0.1% ethanol (v/v), and the third with sterilized double-distilled water
in same volume. The addition of 0.1% DMSO or ethanol did not show
toxic effects on algal growth under the tested growth condition. In
the screening process, we determined the changes of biomass and
astaxanthin content in cells, rst in 24-well cultivation plates coupled
with ow cytometric analysis, and then in 250-mL ask cultivation
with astaxanthin extracted for more precise quantitation. Each chemical
was evaluated at multiple concentrations. The chemical modulators
were applied to H. pluvialis after 3 d green vegetative phase to initiate
the reddish astaxanthin accumulation phase, and the cells were harvested after 9 d when the astaxanthin accumulation reaches late exponential phase (Suppl. Fig. S1).
Oxidant, as reactive oxygen species (ROS), could be used to initiate
production and accumulation of many bioproducts [37]. The results
showed that oxidant, H2O2, AAPH, MB, MV, were found enhancing volumetric productivity of astaxanthin in H. pluvialis by 20.60%, 29.17%,
41.91% and 32.87% at optimal concentration of 0.7 mM, 0.01 mM,
0.001 mM, and 10 7 mM, respectively (Fig. 1). Oxidant treatments
did not show obvious effect on cell density at their optimal concentration (Suppl. Fig. S2), except for H2O2 treatment, which has an agglomerate effect on microalgal cells. Active oxygen generators, Fe2+, MB, AAPH,
MV and H2O2 were capable of increasing astaxanthin accumulation in
H. pluvialis, in which Fe2 + served as an HO generator via an ironcatalyzed Fenton reaction [16,38,39]. Apart from trials in H. pluvialis,
H2O2 was also reported to serve as a chemical trigger to efciently enhance cellular astaxanthin content by 83% in Xanthophyllomyces
dendrorhous[40]. Similarly, Ma and Chen found that Fe2 + (0.5 mM)
added to the medium with H2O2 (0.1 mM) or MV (0.01 mM) promoted
astaxanthin formation to 7.1 mg/g and 6.3 mg/g, respectively in
Chlorococcum sp. [41,42]. The addition of 0.1 mM H2O2 to the culture

also enhanced the yield of astaxanthin from 9.9 mg/L to 12.58 mg/L in
Chlorella zongiensis[43]. Carotenoid formation in algal cyst cells enhanced by HO or other active oxygen species (1O2, O
2 , H2O2, and
AO2) might be related to their roles on participating directly in the
carotenogenic enzyme reactions as an oxidizer or an H acceptor [44].
Effect of ROS also triggers antioxidants, such as glutathione (GSH) and
ascorbic acid (AsA) and antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT) and ascorbic peroxidase (APX) [45].
Signal transducers play a key role in the complicated signaling networks in plant and microalgal defense responses [4648]. In the study,
signal transducers SA, JA, ABA were found to enhance astaxanthin accumulation in H. pluvialis by 30.55%, 50.56% and 64.21% at optimal concentration of 0.724 mM, 0.048 mM, and 0.378 mM, respectively (Fig. 1).
Signal transducer treatments did not show obvious effect on cell density
at their optimal concentration (Suppl. Fig. S2), thus the volumetric productivity enhancement was mainly caused by the increased astaxanthin
content. Among these chemicals, ABA treatment presented the most
signicant stimulatory effect on astaxanthin production (33.6 mg/L),
which is 64.21% higher than its DMSO control. Early study showed
that ABA, as an effective regulator for astaxanthin production in
H. pluvialis, induced a morphological change from green vegetative
cells to red cyst cells [49]. More recently, studies found that chemicals
JA and SA could enhance astaxanthin production to 1.458 mg/L and
2.74 mg/L, respectively [19,50]. In addition, methyl jasmonate (MJ),
the methyl ester of JA, was found with roles in regulating expression
of the bkts gene that catalyzes -carotene to canthaxanthin in the
carotenogenesis pathway [51].
In addition, GA3 and metal ion Mg2+ also enhanced volumetric productivity of astaxanthin accumulation by 30.82%, and 29.87% at their
own optimal effective concentration, respectively (Fig. 1). No difference
in terms of growth was observed for the treatments by GA3 and Mg2+
(Suppl. Fig. S2). GA3 was reported to have a positive role on astaxanthin
biosynthesis in H. pluvialis by regulating carotenoid gene expression [18]. 0.7 g/L Mg2+ was also demonstrated to stimulate astaxanthin
accumulation in Phafa rhodozyma with 0.1 g/L Mn2+ and 0.2 g/L Ca2+.
Under the optimal conditions, the astaxanthin yield reached 2.95 mg/L,
which is 1.31 times of that without metal ions [52].
3.2. Targeted LCMS metabolomic analysis
Considering their signicant stimulatory effects, oxidants and signal
transducers could serve as promising chemical modulators for

Table 1
Effect of chemical modulators in astaxanthin.
Type

Chemical

Optimal concentration (mM)

Effect on Ax %

Antioxidant

Butylated hydroxyanisole (BHA)

Propyl gallate (PG)
H2O2H2O2
2,2-Azo-bis(2-amidinopropane)-dihydrochloride (AAPH)
Methylene blue (MB)
Methyl viologen (MV)
Salicylic acid (SA)
Jasmonic acid (JA)
2,4-Epibrassinolide (EBR)
Abscisic acid (ABA)
MgCl2
ZnSO4
CuSO4
3-Iodoacetate acid (IAA)
3-Indolebutyric acid (IBA)
Naphthoxyacetic acid (BNOA)
1-Naphthaleneacetic acid (NAA)
2,4-Dichlorophenoxy acetic acid (2,4-D)
Gibberellic acid (GA3)
Zeatin (ZA)
Kinetin (KT)
2-Chlorodracylicacid
Ethanolamine (ETA)

/
/
0.7
0.01
0.001
107
0.724
0.048
/
0.378
5
/
/
/
/
/
/
/
0.029
/
/
/
/

/
/
20.60 0.81
29.17 4.38
41.91 1.90
32.87 3.01
30.55 0.73
50.56 4.06
/
64.21 3.33
29.87 5.19

/
/
/
/
/
/
30.82 3.37
/
/
/

Oxidant

Signal transducer

Metal ion

Auxin

Gibberellin
Cytokinin

Amines

X. Yu et al. / Algal Research 11 (2015) 284293

90

80

D) MV

130

0.7 mM

**
*

110
100
90

10-9 mM

G) GA3

150
140

10-7 mM

90

***

120
110
100
90

**

110

90

Control

Ethanol

0.014 mM 0.029 mM 0.058 mM

***

120
110
100
90
80

Control

10-4 mM

10-3 mM

10-2 mM

**

F) Mg

130
120
110
100
90
80

Control

Control

0.362 mM 0.724 mM 1.448 mM

180

**

H) JA

170

150
140

**

130
120
110
100
90
80

80

140

140

**

***

C) MB

130

10-1 mM

100

160

**

130

10-2 mM

E) SA

120

170

10-3 mM

130

80

10-5 mM

volumetric production%

Control

volumetric production%

100

Control

80

160

110

140

120

170

**

120

1.0 mM

volumetric production%

volumetric production%

140

0.5 mM

150

130

80

Control

**
volumetric production%

100

160

B) AAPH

volumetric production%

**

140

volumetric production%

110

150

A) H2O2
*

volumetric production%

volumetric production%

120

287

2 mM

5 mM

10 mM

***

I) ABA

160
150

***

140

**

130
120
110
100
90
80

Control

DMSO

0.024 mM

0.048 mM

0.095 mM

Control

DMSO

0.189 mM 0.378 mM

0.757 mM

Fig. 1. Effect of chemical modulators on volumetric production (mg astaxanthin/L liquid). The volumetric production of astaxanthin in the control was set as 100%, and the % changes under
various concentrations of the chemicals were shown. Statistical analysis was conducted as described in the text, as signicance indicated by *: p b 0.05; **: p b 0.01; ***: p b 0.001. A) H2O2;
B) AAPH;C) MB; D) MV; E) SA; F) Mg; G) GA3; H) JA; I) ABA.

astaxanthin production for large-scale fermentation of H. pluvialis To

investigate stimulatory mechanisms, we employed a LCMS based
metabolomic approach to comparatively analyze cellular metabolism
under various chemical modulators to quantify unstable metabolites,
such as the hydrolytically unstable nucleotides (ATP, GTP, cAMP, and
PEP) and the redox active nucleotides (NADPH, NADH) that are crucial
for major metabolic pathways. In this experiment, we chose three
chemicals from each group of chemical modulators based on their
effects on astaxanthin accumulation, namely AAPH, MB and MV from
the oxidant group, and SA, JA and ABA from the signal transducer
group, respectively, to analyze their effects on cellular metabolism.
These chemicals were added to the cultures at 3 d, and the two controls,
one with 0.1% DMSO and one without, were also established in parallel.
The cells were collected at 6 d and 9 d, which are corresponding to middle and late exponential phases of astaxanthin accumulation, respectively (Suppl. Fig. S1). 24 selected metabolites related to central
carbohydrate and energy metabolism were quantied by the LCMS
based targeted metabolomic protocol in the H. pluvialis cells with and
without chemical modulators [35]. The 24 metabolites covered by the
metabolomic approach were acetyl coenzyme A (Ac-CoA), NADPH,
NADP, NADH, NAD, ATP, ADP, AMP, ADP-GCS, UDP-GCS, coenzyme A
hydrate (CoA), D-fructose 1,6-bisphosphate (FBP), D-fructose 6phosphate (F6P), D-glucose 6-phosphate (G6P), D-ribose 5-phosphate
(R5P), D-ribulose 1,5-bisphosphate (RiBP), D-3-phosphoglyceric acid
(3PG), dihydroxyacetone phosphate (DHAP), DL-glyceraldehyde 3phosphate (GAP), phosphor-(enol)-pyruvic acid (PEP), L-glutamic acid
(GLU), oxaloacetic acid (OXA), -ketoglutaric acid (AKG), and fumarate
dibasic (FUM), most of which are unstable metabolites. At 6 d and 9 d,
these targeted intracellular metabolites in samples were quantitatively

analyzed (Suppl. Table S1). For each sample, three biological replicates
were independently prepared and analyzed.
The results of LCMS metabolomic were presented by the PLS-DA
analysis: i) at both time points, three biological replicates of each sample were clearly clustered together, demonstrating a good reproducibility; ii) the samples treated with or without chemical modulators could
be clearly separated in the PLS-DA plots at both time points, indicating
that various metabolic proles occurred upon chemical modulator and
control treatments; in addition, the results also demonstrated that the
LCMS methodology we applied in the study is highly sensitive to
distinguish the cellular responses upon chemical trigger treatments;
iii) in the plots, at 9 d when astaxanthin accumulation nearly reached
the stationary phase, samples treated with chemical modulators were
separated from the controls, in good agreement with the levels of
astaxanthin accumulation; in addition, between various chemicals,
JA and ABA treatments were the furthest from their control, while
AAPH treatment was very close to the control in the plots, illustrating
the different metabolic changes related to astaxanthin content; iv)
well separation of the metabolomic proles of the samples treated
with signal transducer and oxidant was clearly observed at both 6 d
and 9 d for most of the chemicals, suggesting that these two groups of
chemicals have different and distinct regulative mechanisms on cells.
Overall, the results demonstrated that the LCMS methodology used
in the study provided a high analytical resolution to distinguish samples
treated with various chemical modulators (Fig. 2).
To further illustrate the quantitative information of the metabolic
datasets, heat maps of 24 metabolites in all samples at both 6 d and
9 d were generated (Fig. 2). The metabolites detected by LCMS are
mostly unstable metabolites closely related to energy metabolism and

288

X. Yu et al. / Algal Research 11 (2015) 284293

B) 9 d

A) 6 d
6d

PC2: 25.74%

PC2: 16.49%

-2
C
DMSO
AAPH
MB

-4

-8

-6

-4

-2

MV
SA
JA
ABA

9d

4
2
0
-2
-4
-6

-8

-6

-2

-0.25

0.25

0.25

Control-6 d-1
Control-6 d-2
Control-6 d-3
DMSO-6 d-1
DMSO-6 d-2
DMSO-6 d-3
AAPH-6 d-1
AAPH-6 d-2
AAPH-6 d-3
MB-6 d-1
MB-6 d-2
MB-6 d-3
MV-6 d-1
MV-6 d-2
MV-6 d-3
SA-6 d-1
SA-6 d-2
SA-6 d-3
JA-6 d-1
JA-6 d-2
JA-6 d-3
ABA-6 d-1
ABA-6 d-2
ABA-6 d-3

Control-6 d-1
Control-6 d-2
Control-6 d-3
DMSO-6 d-1
DMSO-6 d-2
DMSO-6 d-3
AAPH-6 d-1
AAPH-6 d-2
AAPH-6 d-3
MB-6 d-1
MB-6 d-2
MB-6 d-3
MV-6 d-1
MV-6 d-2
MV-6 d-3
SA-6 d-1
SA-6 d-2
SA-6 d-3
JA-6 d-1
JA-6 d-2
JA-6 d-3
ABA-6 d-1
ABA-6 d-2
ABA-6 d-3

-0.25

-4

PC1: 30.53%

PC1: 33.69%

DHAP
ATP
UDP-GCS
3PG
PEP
NADPH
AcCOA
NAD
RiBP
ADP-GCS
GAP
FBP
R5P
GLU
NADH
AMP
NADP
ADP
G6P
F6P
COA
OXA
AKG
FUM

ADP-GCS
RiBP
FBP
ATP
AcCOA
NADPH
DHAP
R5P
GAP
PEP
AMP
F6P
ADP
3PG
NADP
G6P
GLU
NAD
UDP-GCS
NADH
COA
OXA
AKG
FUM

Fig. 2. Score plots of the PLS-DA analysis (upper) and heat maps of LCMS metabolomic proles (low). C represents control media, and different colors represent media supplemented with
indicated various chemical modulators. The scattered plots represent biological replicates in PLS-DA. PC1 is principle component one and PC2 is principal component two. The number
represents biological replicates in the heat map. A) 6 d; B) 9 d. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)

central carbohydrate metabolism. Heat map analysis of the metabolic

effects of chemical modulators added alone showed that: i) for signal
transducer, SA, JA and ABA treatments had caused similar responses
that G6P, GLU, OXA, COA and FUM were mostly down-regulated at
6 d, meanwhile G6P, F6P and COA were clearly up-regulated at 9 d. In
addition, R5P, involved in pentose phosphate pathway (PPP), a process
that generates NADPH and pentoses (5-carbon sugars) [53], was clearly
up-regulated at both 6 d and 9 d in SA and JA treatments, meanwhile
NADPH was down-regulated at 9 d, suggesting that increased supply
and consumption of NADPH could be required for fast astaxanthin accumulation. In addition, a previous study has found that NADPH played an
important role in supporting the fungal oxidative system and in maintaining redox balance for the metabolism of aromatic compounds in
Phanerochaete chrysosporium[54]; ii) for oxidant AAPH, MB and MV, it
was found that at 6 d, except for RiBP and NADPH that were upregulated in all samples, very few metabolites showed similar patterns
of regulation. For example, down-regulation of AMP, ADP, 3PG and
NADP, and up-regulation of FBP and NADPH were found in the AAPH
treatment; while in the MB treatment, AcCOA, NADPH and UDPglucose were up-regulated and COA, OXA, AKG and FUM were downregulated. In the MV treatment, up-regulation of ADP-GSC, DHAP, R5P,
GAP and PEP and down-regulation of NADP, G6P, GLU, NAD, NADH,
COA, OXA, AKG and FUM were observed, respectively, suggesting that
different types of oxidants might have different effects on central carbohydrate metabolism at the initial astaxanthin accumulation stage. At
9 d, RiBP was also up-regulated while DHAP, AKG and FUM were
down-regulated in all oxidant treatments. The low concentration of

metabolites in TCA cycle (i.e., AKG and FUM) under oxidative stress is
consistence with a previous study in P. chrysosporium[54]; and iii) between the two groups of chemicals, GAP and R5P were clearly downregulated in AAPH and MB treatments at 6 d, while up-regulated in all
signal transducer treatments suggesting that oxidant and signal transducer might have different regulatory mechanisms to astaxanthin biosynthesis in H. pluvialis. Interestingly, MV and SA, although in different
chemical groups, showed similar metabolic proles at 9 d. In MV and
SA treatments, glycolysis seemed strengthened, with up-regulation of
PEP and G6P, F6P, FBP, and 3PG, respectively, suggesting that MV and
SA might induce astaxanthin by triggering glycolysis process to generate more precursors of carotenoid. Consistently, a previous metabolite
proling of Chlamydomonas reinhardtii under sulfur stress showed that
the concentrations of a number of phosphorylated glycolysis intermediates, G6P, F6P and 3PG increased by 3.7, 3.2 and 2.8 fold, respectively
[55].
3.3. Untargeted GC-MS metabolomics analysis
Using a GCMS metabolomic protocol developed previously [33,34],
we determined the metabolic proles of samples treated with different
chemical modulators. Following the same experimental design as
LCMS analysis described above, cell samples with or without chemical modulators were collected at two time points (i.e., 6 d and 9 d)
and analyzed. For each sample, three biological replicates were independently prepared and analyzed. The GCMS analysis identied 94
chemically classied metabolites from H. pluvialis cells, including a

X. Yu et al. / Algal Research 11 (2015) 284293

large number of compounds such as fatty acids, amino acids, carbohydrate and terpenoids (Suppl. Table S2). Although some of these chemical modulators have been reported in cells, our GCMS metabolomic
analysis detected none of them, suggesting that their intracellular concentration may be very low. In the early studies, a maximum content of
H2O2 was reported to be 5.93 mol/g fresh weight in Brassica juncea
plants under Cd stress treatment [56], and a maximum ABA content in
maize kernels was reported under 350 pg/mg liquid [57]. These concentrations in cells are far less than the chemical modulator concentrations
we used to increase astaxanthin in this study.
The metabolic proles were presented by PLS-DA score plots to
show the similarities and differences between metabolomic proles
(Fig. 3). The features of score plots of the GCMS metabolomic proles
were similar to the LCMS metabolomic proles as we described before,
such as overall good reproducibility between biological replicates and
clear separation between different sample clusters. At 6 d, samples
treated with chemical modulators were separated from the controls,
and the relative distances of the chemical modulator treated samples
from the controls were inconsistent with the levels of astaxanthin accumulation. Moreover, the samples treated with oxidants and signal
transducers were separated from each other except MV, consistent
with the results of LCMS analysis, illustrating that these two groups
of chemical modulators might have distinct effects on cellular metabolism in H. pluvialis. At 9 d, all samples treated with chemical modulators
tend to be clustered together away from the control, which might be
due to the cell aging that caused the similar metabolic responses at
this stage. In addition, the results also demonstrated that the integrated
GCMS and LCMS based metabolomics could encompass a better coverage of both unstable and stable intercellular metabolites as GCMS
based metabolomics can achieve a good coverage of polar metabolites,
such as fatty acids and amino acids, and permit analysis of a wide
range of chemical metabolite classes in a single run [58], while LCMS
can cover unstable metabolites that is incompatible with GCMS
analysis.

PC2: 13.41%

289

3.4. WGCNA correlation network construction

To recognize metabolic modules related to various chemical modulator treatments, a WGCNA network analysis was applied to the GC
MS metabolomic datasets. WGCNA is a correlation-based and unsupervised computational method to describe and visualize correlation
patterns of data points [29], and we have recently applied the method
successfully to metabolomic data of various microbes [11,35,36]. Following the standard protocol, unsigned networks were constructed by
the GCMS metabolomic datasets of both time points, and then localized the correlated metabolites into various metabolic modules; in addition, the association of each distinguished metabolic module with
chemical treatment was also determined, as highly associated modules
indicated on the plots. Setting a minimal number of metabolites in any
module greater than 3, a cutoff of correlation coefcients (r value) between module and treatment condition greater than 0.5 or less than
0.5, and their statistical condence (p-value) of less than 0.05, the
WGCNA analysis identied 3 (named as 6 d-M1, 6 d-M2, 6 d-M3) and
3 (named as 9 d-M1, 9 d-M2, 9 d-M3) distinct metabolic modules at
6 d and 9 d, respectively (Suppl. Figs. S3 and S4). Moduletrait relationships between each chemical modulator treatment and metabolic
module were also presented (Suppl. Figs. S5 and S6). Metabolites associated with each of the responsive modules were provided in Table 2.
Analysis of metabolites within each metabolic module showed signicant differences; for example, 6 d-M1 and 9 d-M3 were composed
mostly of metabolites involved in central carbohydrate metabolism,
such as D-malic acid, fumaric acid and alpha ketoglutaric acid; 6 d-M2
and 9 d-M1 modules included more metabolites involved in fatty acid
metabolism, such as heptadecanoic acid, stearic acid, arachidic acid
and palmitic acid; and 6 d-M3 and 9 d-M2 modules included mostly
amino acid and carbohydrate, such as L-valine, L-glutamic acid, tagatose
and talose. Interestingly, the results showed that oxidants and signal
transducers had opposite associations with module 6 d-M1 and module
9 d-M1. While these results demonstrated that oxidants and signal

A) 6d

-4

-8

-10

-8

-6

-4

-2

10

PC1: 16.28%

PC2: 12.87%

B) 9d

-4

-8

-10

-8

-6

-4

-2

PC1: 16.53%

10

C
C+DMSO
AAPH
MB

MV
SA
JA
ABA

Fig. 3. Score plots of the PLS-DA analysis of GCMS metabolomic proles. C represents control media, and different colors represent conditions supplemented with various chemical modulators as indicated. The scattered plots represent biological replicates. A) 6 d; B) 9 d. PC1 is principal component one and PC2 is principal component two, respectively. (For interpretation
of the references to color in this gure legend, the reader is referred to the web version of this article.)

290

X. Yu et al. / Algal Research 11 (2015) 284293

Table 2
Hub metabolites, member metabolites and pathways clustered into each of responsive modules.a
Modules Phenotype associated Association (r2) p-value

Metabolites

Hub metabolites

Pathway enrichment

6 d-M1

Oxidant
Signal transducer

0.54
0.82

0.006
L-Mimosine, glycerol, porphine, fumaric acid, L-serine, capric acid, D-malic acid, L-pyroglutamic acid, L-cysteine,
8 107 alpha ketoglutaric acid, 1,3-diaminopropane, L-asparagine, putrescine, D-mannose, behenic acid, sucrose,

6 d-M2

Signal transducer

0.5

0.01

6 d-M3

Oxidant

0.51

0.01

9 d-M1

Oxidant
Signal transducer

0.5
0.87

0.01
3 108

9 d-M2

Signal transducer

0.57

0.003

9 d-M3

Oxidant

0.82

9 107

D(+)-trehalose, squalene
Benzoic acid, 2-amino-1-phenylethanol, glycine, uracil, L-threonine, nicotinamide, 1-methyl nicotinamide, citric
acid, tyrosine, isopropyl-beta-D-1-thiogalactopyranoside, palmitic acid, allo inositol, DL-3,4-dihydroxyphenyl
glycol, heptadecanoic acid, stearic acid, arachidic acid, dioctyl phthalate
L-Valine, L-norleucine,

phosphoric acid, L-proline, L-glutamic acid, phenylalanine, pipecolic acid, lauric acid,
dihydroxyacetone phosphate, glycerol-1-phosphate, myristic acid, beta-glycerolphosphate, DL-isoleucine,
maltose, L-ornithine, 3-phosphoglycerate, cellobiose, fructose, adenosine
Urea, succinic acid, D-malic acid, phenylalanine, dihydroxyacetone phosphate, glycerol-1-phosphate,
3-phosphoglycerate, citric acid, D-allose, palmitic acid, cytindine-5-monophosphate, linoleic acid, stearic acid,
arachidic acid
Adenosine-5-monophosphate, squalene, adenosine, allo-inositol, aspartic acid, DL-3,4-dihydroxyphenyl glycol,
D-lyxos, L-(+)

L-Mimosine,

lactic acid, L-alanine, lauric acid, L-valine, malonic acid, sucrose, tagatose, talose

porphine, glyceric acid, fumaric acid, capric acid, adenine, rafnose

GCMS metabolic database was used for this analysis.

transducers could have enhanced astaxanthin accumulation through

different regulatory mechanisms, the details of their own regulations
remained unclear.
Hub metabolites are with a high degree of connectivity in biological
interaction networks and are thus supposed with high biological importance. Assuming hub metabolites with connectivity greater than 5, from
the WGCNA network we were able to identify 4 (fumaric acid, D-malic
acid, arachidic acid and maltose) and 3 (urea, sucrose, and fumaric

acid) hub metabolites signicantly associated with astaxanthin contents at 6 d and 9 d, respectively (Fig. 4). At 6 d, i) in the 6 d-M1 module,
the two hub metabolites, fumaric acid and D-malic acid were identied
and connected to several other metabolites in the module. A derivative
of fumaric acid, fumaric acid-molasses, has been reported previously to
promote biosynthesis of canthaxanthin to 9.2 mg/L in Brevibacterium sp.
strain KY-4313 [59]. Malic acid was reported to have a stimulatory effect
on the formation of astaxanthin in C. zongiensis since it could be

B) 6 d-M2

A) 6 d-M1

Glycine

Putrescine

Arachidic acid

D-malic acid
2-amino-1-phenylethanol

L-pyroglutamic acid

Palmitic acid

Porphine
Stearic acid

Fumaric acid

Heptadecanoic acid

Squalene
Benzoic acid

D-mannose

D) 9 d-M1

C) 6 d-M3

Stearic scid
Phenylalanine

Arachidic acid

Phosphoric acid
Urea
Maltose

L-proline

L-norleucine

Succinic acid

Lauric acid
Citric acid
L-glutamic acid
Cytindine-5-monophosphate

Dihydroxyacetone phosphate

Myristic acid

Linoleic acid
Cellobiose

F) 9 d-M3

E) 9 d-M2
Sucrose

Porphine

D-lyxose

Raffinose
Tagatose

Adenosine

Maltotriose
Fumaric acid

Allo-inositol

L-alanine

Lauric acid

DL-3,4-dihydroxyphenyl glycol

L-mimosine
Glyceric acid
Adenine

Fig. 4. Hub metabolites and their metabolic proles as represented by node and edge graph. A) D-Malic acid and fumaric acid for 6 d-M1; B) arachidic acid for 6 d-M2; C) maltose
for 6 d-M3; D) urea for 9 d-M1; E) sucrose for 9 d-M2; F) fumaric acid for 9 d-M3. Only those nodes with high connectivity strength as displayed near the edges were shown. Hub
metabolites were marked in bold.

X. Yu et al. / Algal Research 11 (2015) 284293

converted to pyruvate, which might then serve as a precursor for IPP, a

carotenoid precursor in C. zongiensis and H. pluvialis[60]. Additionally,
squalene in the same module was reported to have a negative effect on
echinenone and trans-astaxanthin content while -carotene concentration remained constant, suggesting that squalene would specically inhibit the conversion of -carotene into echinenone in the metabolic
pathway of astaxanthin synthesis [50] (Fig. 4A); ii) in the 6 d-M2 module, the hub metabolite arachidic acid was connected to palmitic acid,
heptadecanoic acid, benzoic acid, 2-amino-1-phenylethanol and glycine. Previous fatty acid prole analysis showed that the relative content
of palmitic acid (C16: 0), heptadecanoic acid (C17: 0), stearic acid (C18:
0), arachidic acid (C20: 0) and linoleic acid (C18: 2n6) were regulated
by different stress conditions in H. pluvialis[61,62]. In addition, the
derivative of arachidic acid, hydroxylated eicosanoids, was also found
involved in wound response of the red alga Gracilaria chilensis[63], suggesting that fatty acid concentration might very under chemical stress
and correlated to astaxanthin biosynthesis in this study (Fig. 4B);
iii) in the 6 d-M3 module, the hub metabolite, maltose, was identied.
In our previous study, the isomeride structure of maltose, isomaltose was reported to be negatively associated with increased lipid accumulation caused by butylated hydroxyanisole (BHA) treatment in
Crypthecodinium cohnii [54]. Among the metabolites linked to maltose
in the module, glutamic acid was previously found associated with
astaxanthin-induced Fe2 +and high-light condition in H. pluvialis[11]
(Fig. 4C). At 9 d, i) the hub metabolite of the 9 d-M1 module, urea was
connected to fatty acids, succinic acid and citric acid. Although never
reported in H. pluvialis, citric acid and succinic acid have been found to
decrease in P. chrysosporium exposed to exogenous benzoic acid [51]
(Fig. 4D); ii) in the 9 d-M2 module, the hub metabolite, sucrose was
identied (Fig. 4E). Sucrose was reported as an efcient carbon resource
in C. zongiensis for astaxanthin production [64], suggesting that intracellular sucrose might be related closely with astaxnthin biosynthesis;
iii) in the 9 d-M3 module, fumaric acid was identied as a hub metabolite with rafnose, porphine, maltotriose, glyceric acid and adenine connected to it in the module (Fig. 4F). Rafnose was reported to serve as
scavenge hydroxyl radicals to protect plant cells from oxidative damage
caused by MV treatment, salinity, or chilling in leaves of Arabidopsis[65],
and glyceric acid was involved in resistance to drought stress in grapevine leaves [66]. Overall, although it needs further conrmation, the hub
metabolites and their associated metabolites detected in this analysis
might serve as biomarkers of astaxanthin biosynthesis in H. pluvialis.
3.5. Pathway enrichment
Pathway enrichment analysis of the metabolites in the responsive modules was also conducted and the statistical signicance
(i.e., p-values) was determined (Table 2). Using the pathways with at
least two of its member metabolites identied, the enrichment analysis
showed that one pathway (Glutathione metabolism) for the 6 d-M1
module, one pathway (Biosynthesis of unsaturated fatty acids) for
the 6 d-M2 module, two pathways (ABC transporters and Tropane,
piperidine and pyridine alkaloid biosynthesis) for the 6 d-M3 module,
four pathways (Glycerophospholipid metabolism, Glycerolipid metabolism, Biosynthesis of unsaturated fatty acids, and Biosynthesis
of phenylpropanoids) for the 9 d-M1 module, and one pathway
(Butanoate metabolism) for the 9 d-M3 module, were identied
with statistical signicance p-values of less than 0.05. However, no signicant pathway enrichment was found for module 9 d-M2. Glutathione (GSH) in Glutathione metabolism, similar to astaxanthin, is an
important antioxidant that is necessary to prevent oxidative injury to
cyst cells caused by exposure to reactive oxygen in H. pluvialis[7,67].
Additionally, the cellular contents of ascorbic acid and GSH in Spirulina
platensis grown under 8 mM H2O2 stress were up-regulated 4.3- and
3.0-fold, respectively, when compared with the control [43]. As for
Biosynthesis of unsaturated fatty acids pathway, active synthesis of
fatty acids has been found coordinated with astaxanthin production,

291

as cerulenin, a specic inhibitor of fatty acid synthesis, also inhibited

astaxanthin accumulation [31]. In addition, a linear correlation was
also found between the increases in the cellular content of astaxanthin
and oleic acid under nitrogen starvation condition [68].
4. Conclusions
Chemical modulators that regulate cellular metabolism to enhance
accumulation of bioproducts could be an economical and effective approach to increase productivity in the microalgae-based biotechnology
application. To identify new chemical modulators, we screened 23
chemical modulators from eight functional groups and were able to
nd that chemicals of oxidant and signal transducer groups had signicant stimulatory effects on astaxanthin accumulation in H. pluvialis. To
further investigate the enhancing mechanism, we employed an integrated LCMS and GCMS metabolomic approach to determine the
metabolic changes in vivo treated with 6 chemicals from the oxidant
and signal transducer groups, and further network analysis uncovered
6 distinguished metabolic modules and 7 hub metabolites highly associated with the treatments of signal transducers and oxidants. In addition
to identication of several chemical modulators that may have practical
application in astaxanthin accumulation in H. pluvialis, the study provided a metabolomic characterization of the regulatory mechanisms of
oxidants and signal transducers on astaxanthin accumulation in
H. pluvialis.
Conict of interest
The authors have no conict of interest to declare.
Authors' contributions
LC and WZ conceived the design of the study, XY carried out the
induction experiment, XY, XZ and JL performed the astaxanthin quantication, XY, XN and XZ performed GC and LC sample preparation, XY
and GP performed the GCMS analysis, XY and XZ performed the
LCMS analysis, and XY, LC and WZ drafted the manuscript. All authors
read and approved the nal manuscript.
Abbreviations
2,4-D
3PG
AAPH
ABA
Ac-CoA
AKG
AsA
ATCC
BHA
BNOA
CAT
CoA
DHA
DHAP
DMSO
EBR
EMP
ETA
F6P
FBA
FBP
FUM
G6P
GA3
GAP

2,4-Dichlorophenoxy acetic acid

D-3-phosphoglyceric acid
2,2-Azo-bis(2-amidinopropane)-dihydrochloride
abscisic acid
acetyl coenzyme A
-Ketoglutaric acid
ascorbic acid
American Type Culture Collection
butylated hydroxyanisole
naphthoxy acetic acid
catalase
coenzyme A hydrate
docosahexaenoic acid
dihydroxyacetone phosphate
dimethyl sulfoxide
2,4-Epibrassinolide
EmbdenMeyerhof pathway
ethanolamine
D-Fructose 6-phosphate
fructose-1,6-bisphosphate aldolase
D-Fructose 1,6-bisphosphate
fumarate dibasic
D-Glucose 6-phosphate
gibberellic acid
DL-glyceraldehyde 3-phosphate

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X. Yu et al. / Algal Research 11 (2015) 284293

GCMS gas chromatographymass spectrometry

L-Glutamic acid
GLU
GSH
glutathione
IAA
3-Iodoacetate acid
IBA
3-Indolebutyric acid
JA
jasmonic acid
KT
kinetin
LCMS liquid chromatographymass spectrometry
MB
methylene blue
MDH
malate dehydrogenase
MV
methyl viologen
NAA
1-Naphthaleneacetic acid
OXA
oxaloacetic acid
PLS-DA partial least squares-discriminate analysis
PEP
phosphor-(enol)-pyruvic acid
PG
propyl gallate
PPP
pentose phosphate pathway
D-Ribose 5-phosphate
R5P
D-Ribulose 1,5-bisphosphate
RiBP
ROS
reactive oxygen species
SA
salicylic acid
SOD
superoxide dismutase
TCA cycle tricarboxylic acid cycle
UDP-GCS uridine 5-diphosphoglucose
ZA
zeatin
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.algal.2015.07.006.

Acknowledgments
This study was supported by the funds from the National High-tech
RD Program (National 863 program) (No. 2012AA02A707), the National Basic Research Program of China (National 973 program) (No.
2014CB745101, No. 2012CB721101 and No. 2011CBA00803), the Doctoral Program of Higher Education of China (No. 20120032110020 and
No. 20130032120022), and the Tianjin Municipal Science and Technology Commission.

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